Cyclic GMP-dependent protein kinase (PKG) plays a central role in the regulation of vascular smooth muscle tone. Activation of PKG leads to alterations in intracellular Ca2+, which in turn, effects multiple cellular signaling pathways. However, progress in understanding the specific functional roles of PKG in vascular smooth muscle has been hampered by the lack of specific PKG inhibitors. We have developed novel, cell-permeable PKG specific peptide inhibitors with unprecedented potency and kinase specificity by fusion of peptides derived from combinatorial libraries with membrane translocation signal (MTS) peptides. These fusion peptides result in an extraordinary synergism with respect to PKG inhibition. In the proposed experiments, these PKG selective inhibitors will be further developed, refined and used to study specific functional roles of PKG in vascular smooth muscle. Specific Aim 1 will develop novel peptide library design strategies based on binding- and competition libraries. This approach should lead to new and mole potent PKG inhibitors than are currently available. Specific Aim 2 will explore variations of the MTS sequences with respect to their use for intracellular delivery and their ability to synergize with the library derived peptide inhibitors. Specific Aim 3 will determine the contributions of PKG in the regulation of vascular tone in intact resistance arteries by measuring the effects of PKG inhibitors on arterial diameter as well as ion channel activity in single vascular smooth muscle cells. To pursue the aims of this proposal, a multi-faceted approach will be used, including state-of-the-art techniques to: 1) screen and synthesize selective PKG inhibitor peptides using combinatorial library approaches, 2) deliver peptide inhibitors using membrane translocation peptide sequences and monitor the intracellular accumulation of these compounds using kinase activity assays, fluorescence spectroscopy and confocal microscopy, and 3) assess the efficacy of the PKG inhibitors using functional assays of ion channel activity (patch clamp) and vascular contractility (myography). The studies outlined above will provide new, selective, and potent inhibitors of PKG which will be useful in revealing the fundamental physiological roles of PKG in regulation of arterial tone. The application of the inhibitors that emerge from these studies will not only demonstrate the essential role of PKG in regulation of vascular tone in resistance arteries, but also significantly advance the field of protein kinase signaling in general. This work may also suggest novel targets for therapeutic interventions in vascular diseases such as hypertension and septic shock.